This is the first genome-wide analysis on the fasting level of GH that has been conducted, and the largest genetic study on the fasting level of GH in all categories. A GWAS-approach with a discovery phase in 4134 individuals and subsequent replication in a non-overlapping cohort of 5262 individuals identified one SNP on chromosome 17 in which the minor allele was associated with a reduced fasting level of hs-GH. A candidate gene approach identified SNPs in the GHR associated with the fasting level of hs-GH, which in a later phase showed strong associations to height. The main clinical implication of our findings is that genetic signals, such as those identified in the current study, are necessary to be able to assess whether the previously reported associations between high circulating GH levels in the fasted state and risk of cardiovascular morbidity and mortality are causal or not in Mendelian Randomization studies. Also, the novel locus associated with the fasting level of GH represents a completely new biological mechanism behind control of plasma GH level that deserves further study.
The minor allele of rs7208736 was associated with a significant reduction of the fasting level of hs-GH both in the discovery and replication cohort. The results were stronger in females in MDC-CC, but stronger in males in MPP. It should be noted that there were approximately half as many women in MPP as in MDC-CC, which may explain part of the reduced performance among females in MPP. Although the minor allele was associated with a reduction of hs-GH, it was also associated with a lower BMI and waist in the male part of MDC, which may seem counter intuitive since these variables are inversely related to the fasting level of hs-GH [2, 18]. An augmenting modification of the effect of hs-GH could explain the results, since this would lead to larger effects on target tissues and reduced secretion due to enhanced feedback signaling, i.e. the opposite of GH-insensitivity syndrome [21, 22]. It should however be noted that the associations between rs7208736 and BMI and waist are rather weak for such a big cohort, which underlines the need for replication of our results, especially concerning the metabolic effects of this SNP.
Rs7208736 is located in a non-coding sequence of the short arm of chromosome 17 (17p13.3). The GH-gene cluster is located on the same chromosome, but on the other arm, so any direct interaction with this gene is unlikely. However, numerous genes, which the SNP might affect through enhancers or regulatory motifs, are located nearby. Inactivation of the region around rs7208736 has been linked to several malignancies, such as lung, breast, liver, colon, kidney, and brain, with the genes HIC1 (Hypermethylated in cancer 1) and OVCA2 (Ovarian cancer-associated gene 2) being potential tumor suppressors [23, 24]. HIC1 is located approximately 150 kbp downstream of rs7208736, and could be the link between rs7208736 and GH. Among other effects, HIC1 represses transcription of SIRT1 (Sirtuin 1) [23, 25]. SIRT1 is a deacetylase that is activated by caloric restriction and in vivo in rats have been shown to negatively regulate GH-dependent IGF-1 production in the liver by deacetylation of STAT5 [26]. STAT5 is a downstream target of GHR-signaling via JAK2-activation [11]. Thus higher levels of HIC1 would inhibit transcription of SIRT1, which could augment the effect of GHR-signaling and would explain the picture with the minor allele in rs7208736 reducing levels of hs-GH but at the same time also being associated with lower BMI and waist. However this is very speculative, but nevertheless provides a plausible mechanism with support in the literature. The associations are intriguing and further studies will be needed to replicate this finding and determine the underlying pathways.
When investigated in Cardiogram the major allele of rs7208736, which is associated with a higher level of hs-GH, was associated with an OR of 1.05 per major allele for incident coronary artery disease. This is in line with previously published results of higher levels of hs-GH being associated to cardiovascular disease [2, 3]. However one could argue that it does not fit with the suggested theory in the previous section, i.e. the minor allele being associated with augmented sensitivity in the GHR-signaling. It should be noted that Cardiogram included more individuals than MDC, which may suggest that our proposed mechanism in with the minor allele of rs7208736 augmenting GH-signaling may be erroneous. Alternately you could interpret the results as reduced GH-signalling being associated with CVD, and our previous findings being explained as a compensatory mechanism [2]. This emphasizes the need for replication and further studies on the subject.
In the candidate gene approach the minor allele in rs13153388 was associated with a higher hs-GH and lower height. Height is as expected positively associated with hs-GH [18]. If the SNP is associated with a reduced function of the GHR, the consequence could be a mild form of GH-insensitivity syndrome [21, 22], with a reduced effect of hs-GH, i.e. short stature, but, due to reduced feedback signaling, still high levels of hs-GH in plasma. The effect of the SNP on adult height is small, but evident (Fig. 2).
Unfortunately, the results in the GHR-gene did not replicate in MPP. This may be attributable to differences in the composition of the cohorts. When comparing the baseline characteristics between MPP and MDC-CC, the population in MPP is older, consists of more males and a larger proportion are on medications that potentially could affect GH-levels. The fasting levels of hs-GH display a more pronounced gender difference in MDC-CC than in MPP. Notably the reduced difference in MPP is attributable to both an increase in males and a decrease in females. The gender difference in the fasting levels has previously been described to decline with age [27]. Among females, menopausal status and hormone replacement therapy might also affect the decline in hs-GH, since estrogen is positively associated with hs-GH [28]. It may be that these confounding effects have diluted the differences in GH-levels and made associations harder to capture. However there is indication of more long-term effects of GH in the anthropometric analyses with the strong connection to height, which is heritable to about 80% [29], implying that the SNP might affect the levels of GH earlier in life.
The study has some limitations. There was a fair amount of non-responders to the invitations, which may have biased selection of a more healthy population. Also, in MPP, roughly half the participants were excluded before the random selection, since they participated in the MDC (Additional file 1: Table S1). This was necessary to obtain independent cohorts, but also may have biased MPP-cohort, since willingness to participate in studies (i.e. likelihood to participate in both studies) infers a positive health selection, which could reduce the healthiest part of the cohort in the current study. The pulsatile secretion of hs-GH make measurements more difficult, but as previously discussed we have tried to eliminate this issue by drawing blood in the morning hours when the levels are more stable [2, 6,7,8]. It is inevitable that samples are taken from some individuals during a peak, but given the large number of participants it is still possible to analyze hs-GH on a group level. A single fasting value of hs-GH is not a standard clinical test. The correlation between fasting GH and 24-h GH secretion is strong, but with large variability [9]. However, on a population basis, a single fasting value of hs-GH strongly and independently predicts cardiovascular disease and death as we have previously shown [2], which calls for further research on the subject.
As discussed previously there were some differences in the characteristics between the MDC-CC and MPP. However this should be an advantage to evaluate if the results are applicable to a broader population. A concern could be that the individuals in MPP were old and that the function of their GH-IGF-1-axis may have been reduced.
In the GWAS-approach we did not reach genome-wide significance in our analysis, which means that the results have to be interpreted cautiously. However we managed to replicate our findings in a separate cohort, which increases the credibility of the results. This underlines the need of further replication of our results in independent cohorts. Since the genetic variant is not confirmed it is difficult to assess any causality of GH in cardiovascular disease through a Mendelian randomization.
In the candidate gene approach 32 SNPs were included in the initial analysis, a bonferroni-corrected p-value would be P = 0.002 (0.05/32), which the SNP rs13153388 misses by a fine margin. This emphasizes the need for further replication.
Both cohorts (MDC and MPP) are from the same geographic area with the same genetic background and allele frequencies of the studied SNPs were similar in all cohorts. This makes the results difficult to extrapolate to other ethnic groups. The results will need to be replicated in larger studies, as well as in populations with another genetic background to attain a larger generalizability.